SUMMARY Cancer cells typically exhibit genetic instability and accumulate thousands to hundreds of thousands of mutations in their genome. Replicative DNA polymerases are responsible for copying the vast majority of nuclear DNA. Conditions that reduce their ability to accurately and efficiently synthesize daughter DNA molecules can contribute directly to this increased mutagenesis. Recently, heterozygous missense mutations were identified in the exonuclease domain of DNA Polymerase (Pol) ? from several tumor types. These tumors contain the highest number of somatic mutations identified in tumor genomes to date. Despite these significant consequences, the mechanisms that drive this mutagenesis and how this ultimately affects tumorigenesis remain poorly understood. Here we provide evidence that different cancer-associated mutations in human DNA polymerase (Pol) ?, a major replicative DNA polymerase, impair proofreading activity to dramatically different degrees. We further provide evidence that the degree of proofreading impairment corresponds to the level of cellular mutagenesis. The main goal of this project is to test our central hypothesis that observed tumor ultramutator phenotypes result mainly from the suppression of intrinsic proofreading caused by cancer-derived mutations in Pol ? . This hypothesis is based on our preliminary data. Specifically, this project will 1) Establish a kinetic basis for cancer-causing Pol ? mutant alleles; 2) Define the mechanisms through which Pol ? exonuclease domain mutations (EDMs) generate their unique mutational signatures using novel gene-edited cell lines; and 3) Determine the mechanisms through which Pol ? variants contribute to tumor development. The proposed research is innovative due to the multidisciplinary approach that combines in vitro studies with studies using novel engineered human cell lines and mouse models to characterize the effects of cancer- associated Pol ? mutations on genome stability, mutagenesis and tumorigenesis. The novel insights into how defects at the replication fork can influence genomic alterations are also innovative. This contribution is significant because it will provide new and detailed insights into the biochemical mechanisms of how replicative DNA polymerases normally prevent the acquisition of the complex diversity of mutations found in cancer genomes, as well as provide insights into the fundamental mechanisms of DNA replication. This knowledge will deepen our understanding of cancer development and can ultimately serve to inform future studies designed to modulate DNA polymerase activities toward the goal of novel cancer therapeutic strategies.